It is well known that certain enzymes isomerize aqueous aldose monosaccharides to ketose monosaccharides and vice versa. Within recent years, isomerases have attracted considerable commercial interest in glucose syrup isomerization reactions. Such enzymes are frequently referred to by the trade as glucose isomerases.
A typical commercial glucose isomerization process results in an isomerized glucose syrup product containing approximately 40-50% fructose and 50-60% glucose. The glucose isomerization process normally requires from about 24 to 72 hours and will be conducted at temperatures above 50.degree. C. (usually between 60.degree. to 70.degree. C.). Achieving and maintaining sufficient isomerase activity throughout the isomerization process is necessary to obtain acceptable fructose yields.
Isomerases are inherently susceptible to deactivation. In batch processes, reduced isomerase activity is normally compensated by charging the reactor with sufficient isomerase to complete the isomerization reaction. In a continuous isomerization process, isomerase deactivation may be partially corrected by periodic addition of fresh isomerase. Isomerase deactivation creates difficulties and additional expense in the manufacture of fructose syrups.
Illustrative reported glucose isomerases include those derived from organisms of the Acetobacter genus, (e.g., A. Aceti IFO 3282, A. rubiginosus IFO 3243, A. suboxydans NRRL B 72); Aerobacter genus, (e.g., A. strain HN-56, A. cloacae KN-69, A. cloacae NRC 491 and NRC 492); B. stearothermophilus genus (e.g., ATCC 31265, NRRL B-3680, NRRL B-3681 and NRRL B-3682); Bacillus genus (e.g., B. coagulans HN-68, NRRL B-5350 and NRRL B-5351, B. megaterium ATCC 15450, B, fructosus ATCC 15451, etc.); Arthrobacter genus (e.g., A. Sp. IFO 3576, 3580, 3585, 3591, 3601, 3604, A. nov. sp. NRRL B-3724, 3725, 3727 and 3728); Brevibacterium genus (e.g., B. pentoso-aminoacidicum, B. Lipolyticum IFO 3633); Coryne bacterium (Sp. IFO 3597, IFO 3606, IFO 3618, IFO 3697); Escherichia intermedia HN-500; Lactobacillus genus (e.g., L. brevis, L. fermenti, L. gayoni, L. mannitopoeus, L. pentoaceticus); Leuconostoc mesenteroides; Micrococcus genus (e.g., M. rubens ATCC 186, M. varians ATCC 399); Mycobacterium sp IFO 3603 and IFO 3611; Mycoccocus Sp. IFO 3583; Pseudomonas genus (e.g., P. fluorescens, P. boreopolis, P. coronafaciens, P. striafaciens, P. syncyanea, P. synxantha, P tabaci); Nocardie genus (e.g., N. asteroides, N. dassonvillei); Micromonospora coerulea; Microbispora rosea; Microellobosporia flavea; Serratia plymuthica; Streptomycetes genus (e.g., S. sp ATCC 21175, 21176, S. achromogenes, S. albus YT-4, S. albus YT-51, S. aureus, S. bobiliae, S. marcesens, S. californicus, S. coelicolor, S. diastaticus, S. echinatus, S. flavovirens, S. fradiae, S. fulvissimus, S. galilaeus, S. gedamemsis, S. griseolus, S. horbaricolor, S. nygroscopicus S. lipmanii, S. niveoruber, S. olivaceus NRRL B-3583, S. olivochromogenes ATCC 21114, S. phaeochromogenes, S. rochei, S. roseochromogenes, S. rutgerensis, S. tendae, S. venaceus, S. venezuelae ATCC 21113, S. virginiae, S. viridochromogenes, S. wedmorensis, etc.); Actinoplanes genus (e.g., A missouriensis, A. philippinensis, A. armeniacus); etc.
Continuous fixed-bed reactors are primarily employed to produce fructose syrups. In continuous reactors, the desired fructose level is typically obtained by permitting a high glucose containing syrup to flow through a bed of immobilized glucose isomerase or a series of reactor beds until the desired fructose yield is achieved. Fructose productivity by a fixed bed reactor is directly proportional to the isomerase activity. Decreased yields inherently arise because the isomerase deactivates. Ultimately the isomerase deteriorates and becomes totally ineffective and requires recharging with fresh isomerase.
It is well known that isomerases are less susceptible to deactivation when the isomerization reaction is conducted in the presence of one or more metal ion activators. Such activators stabilize and activate the isomerase. It is conventional to incorporate these metal activators into the dextrose feed syrup. The metal ion activators and the requirements will vary and depend upon the isomerase type. When an isomerase is isolated from a new source or in a different form, it is conventional to establish its metal ion activator requirements. Suppliers of commercial isomerases customarily provide technical information with respect to its metal ion activator requirements for a glucose isomerization process.
The activating and stabilizing effect of cobalt, magnesium and manganese upon isomerases in the isomerization reaction of dextrose to fructose have been extensively reported by numerous researchers (e.g., see Tsumura et al., Agr. Biol. Chem., Vol. 29, No. 12, p. 1129-1134, 1965; Yamanaka, Agr. Biol. Chem., Vol. 27, No. 4, p. 265-270, 1963: Takasaki et al., Agr. Biol. Chem., Vol. 33, No. 11, p. 1527-1534, 1969; S. Yoshimura et al., Agr. Biol. Chem., Vol. 30, No. 10, p. 1015-1023, 1966; Danno et al., Vol. 31, No. 3, p. 284-292, 1967; Natake et al., Agr. Biol. Chem. Vol. 28, No. 8, p. 510-516, 1964; Tz-Yuan et al., Sheng Hua Hsuah Pao 4, (3), p. 342-350, 1964; Tsumura et al., Vol. 29, No. 12, p. 1123-1128, 1965; Sato, Dempunto Gijutsu Kenkyu Kaiho, No. 32, p. 81-88, 1965; Fratzke, National Science Foundation Report No. NS-RA-T-74-099, 1974). Cobalt ions in combination with either magnesium or manganese ions are reportedly very effective stabilizers and activators for most glucose isomerases. Calcium and nickel ions in combination with either manganese or magnesium ions have been proposed as isomerase activators (e.g., see Japanese Patent Appln. NS 112591/76). The use and safety of cobalt ions in the production of food grade products is dubious. The growth promoting effects of iron in the culturing of organisms and isomerase production have been recognized. Most studies pertaining to the effect of iron ions upon isomerase activity tend to show it is generally ineffective, especially when compared to either cobalt, manganese or magnesium ions and combinations thereof. An alternative means for producing food grade fructose without necessitating cobaltous ions and yet provide an acceptable level of fructose productivity and isomerase stability would be a desirable goal.
It has been reported that certain isomerases are activated by conducting the isomerization reaction in the presence of thiol activating reagents such as glutathione and cysteine (e.g., see J. Agri. Chem. Soc., Japan 36, No. 12, p. 1013-1016, 1962 by Y. Takasaki et al.; J. Biol. Chem. 218, p. 535, 1956, by M. J. Palleroni et al.; J. Am. Chem. Soc. 77, p. 1663, 1955 by M. W. Slein; and Agr. Biol. Chem., Vol. 28, No. 8, p. 510-516, 1964 by M. Natake et al.). Researchers have also reported sulfhydryl binding agents which react with sulfhydryl groups (e.g., cuprous ions such as cuprous sulphate or chloride, p-chloro-mercuribenzoate, monoiodoacetate, mercurous ions, zinc sulphate, etc.) will inhibit or destroy glucose isomerase activity. Conducting the isomerization reactions in the presence of oxidizing agents, including nascent oxygen, reportedly have an inhibitory effect upon glucose isomerase activity.